Alternative splicing is a key process in the control of mammalian gene expression and a major source of protein diversity. Errors in splicing regulation are implicated in many disease processes, including cancer and inherited disorders of the neuromuscular systems. However, the cellular circuits that control splicing regulation are mostly unknown. New methods that measure splicing changes on a genome-wide scale make possible the discovery of coordinately regulated networks of alternative splicing. The elucidation of the regulatory events underlying this coordinate control will be essential for understanding how groups of exons are controlled during development and disease. This project will support the continued development and dispersal of parallel technologies for measuring alternative splicing initiated by the Black, Fu and Ares labs through prior R24 funding. In the initial project period, several different approaches were developed. Most notably, two splicing- sensitive microarrays, one for mouse and one for human cells, each measuring splicing of about 1300 alternative splicing events in about 1000 genes, were successfully designed, printed and used to capture and analyze data. These arrays were applied to a diverse set of experiments and were successful in uncovering several systems of coordinate splicing control important in cellular differentiation and homeostasis. We propose to continue this productive collaboration with the following aims: (1) We will continue to apply the arrays and analysis methods produced during the previous funding period to questions of splicing regulation, and we will expand their use to additional laboratories studying splicing; (2) we will improve the design and analysis of splicing-sensitive arrays to make them more comprehensive, and reliable, as well as more widely available; and (3) we will develop a promising new approach to genome-wide splicing analysis using high density sequencing methods. This project will broaden the study of splicing regulation to the level of the whole genome, allowing the integration of specific splicing regulatory pathways into our understanding of gene regulation and genome function. PUBLIC HEALTH RELEVANCE Many human diseases, including both cancer and inherited diseases of the neuromuscular systems, are caused by alterations in gene function through a process called alternative pre-mRNA splicing. Although individual changes in splicing have been linked to particular disorders, it is not well understood how programs of splicing affect the larger biology of the cell, and hence how abnormalities in these programs lead to disease. This project will extend our work on methods for examining splicing regulation on a genome wide scale that will allow elucidation of these larger programs of genetic change in disease.